Post-translational modifications are chemical changes to proteins that occur after the primary structure of the protein has been completed via translation. Post-translational modifications include, but are not limited to, phosphorylation, dephosphorylation, proteolysis, protein ligation, glycosylation, sulfation, methylation, and ubiquitination.
Post-translational modifications influence protein behavior. For example, insulin is formed by the post-translational modification of proinsulin, which itself is formed by the post-translational modification of preproinsulin. The post-translational addition or removal of phosphorus from proteins plays a regulatory role in many biochemical pathways and signal transduction pathways.
The analysis of post-translational modifications often requires labor intensive sample preparation and expensive or hazardous chemical reagents such as radioactive materials. For example, protein kinase assays often include the use of radioactively labeled ATP as phosphate donor to a substrate peptide or protein. Following the kinase reaction, the substrate is separated from unreacted radioactive ATP. Any radioactivity incorporated into the substrate is measured, such as by scintillation counting. This assay has drawbacks. The assay generates radioactive waste. Radioactive phosphorus has a short half life, so fresh reagent must be frequently acquired. The assay requires at least micromolar concentrations of ATP, which is one thousand times greater than the millimolar biological concentration of ATP. The concentration of substrate in the assay is often much higher than expected substrate concentrations in vivo.
There remains a need for simpler methods for measuring post-translational modification. The present invention is designed to address that need.